Chromium (III) alpha amino acid complexes

ABSTRACT

Chromium (III) 1:3 complexes of alpha amino acids such as methionine are used as animal nutritional supplements. The chromium ion is complexed with three Methionine molecules to form the coordination complex anion [CR(met) 3   + ]. The complex is easily absorbed to provide a bioavailable source for methionine, chromium and for nutritional supplementation of animals.

FIELD OF THE INVENTION

Bioavailable chromium complexes for use in animal nutrition.

BACKGROUND OF THE INVENTION

The essential role of chromium in nutrition was first recognized bySchwarz and Mertz in 1959 (Schwarz, K. and Mertz, W., “Chromium (III)and the glucose tolerance factor.” Archs Biochem. Biophys. 85:292(1959)). These researchers observed that rats fed torula yeast developedglucose intolerance. However, rats fed brewer's yeast did not developthis condition. A substance present in the brewer's yeast, but not intorula yeast was termed glucose tolerance factor (GTF). Later it wasdemonstrated that the active ingredient in GTF is chromium (III). Sincethese early observations, numerous avenues of research were initiated tobetter understand the nutritional role of chromium. Although much is nowknown about the role of chromium in human and animal nutrition, there ismuch that is not known and many of the effects of chromium in humandisease are still controversial and not well documented. Recently,several reviews have been published that summarize the current state ofknowledge regarding the role of chromium in nutrition. (“Chromium as aSupplement”, Henry C. Lukaski, Ann Rev Nutr. 19:279(1999); “Chromium,Glucose Intolerance and Diabetes”, Richard A. Anderson, Journal of theAmerican College of Nutrition, 17, 548(1998); “The Biochemistry ofChromium”, John B. Vincent, J. Nutr. 130: 715(2000); “Quest for theMolecular Mechanism of Chromium Action and its Relationship toDiabetes”, John B. Vincent, Nutrition Reviews, 58: 67(2000))

The exact nature of the Glucose Tolerance Factor originally proposed in1959 remains elusive. A chromium-containing material that potentiatedglucose metabolism was partially purified from acid-hydrolyzed Brewer'syeast and porcine kidney. The material from yeast received the mostattention and was commonly referred to as yeast GTF. It was reportedthat chromium in yeast GTF was absorbed more readily than inorganicchromium sources. Further, it was proposed that yeast GTF is composed ofchromium (III) ions, nicotinic acid, glycine, glutamic acid andcysteine. (“Preparation of chromium-containing material of glucosetolerance factor activity from Brewer's yeast extracts and by synthesis,E. W. Toepfer”, W. Mertz, M. M. Polansky et al., J. Agric Food Chem,25:162(1977)) The proposed composition of the yeast GTF remainscontroversial and its isolation was not reproducible in otherlaboratories. Additionally, it has been proposed that the isolated yeastGTF may be an artifact produced by acid hydrolysis of special chromiumbinding proteins. (“Is glucose tolerance factor an artifact produced byacid hydrolysis of low-molecular-weight, chromium binding substance?” K.H. Sumrall and J. B. Vincent, Polyhedron, 16: 4171(1997)).

Recently, some progress was made towards understanding the molecularbasis of the action of chromium in regulating carbohydrate and lipidmetabolism. A peptide known as low-molecular-weight chromium-bindingsubstance (LMWCr) has been isolated and is believed to play a criticalrole in modulating the action of insulin on its receptors. This peptideappears to be widely distributed in mammalian tissues and has beenisolated from a number of sources. LMWCr is composed of glycine,cysteine, glutamic acid and aspartic acid. Glutamic and aspartic acidsrepresent more than half the amino acid residues. The peptide is 1500Dalton and binds four chromium ions. It is present in tissues primarilyin its metal-free form. The amino acid sequence of this protein and thecrystal structure of its complex with chromium are not yet known. (“TheBiochemistry of Chromium”, J. B. Vincent, J. Nutr. 130:715(2000)) Itappears that LMWCr-bound chromium is present primarily in the form ofanion bridged multinuclear chromium-carboxylate assembly. (“SyntheticModels for Low-Molecular-Weight Chromium-Binding Substance: Synthesisand characterization of Oxo-Bridged Tetranuclear Chromium (III)Assemblies”, Truitt Ellis et al, Inorg. Chem., 33: 5522(1994)) Asynthetic multinuclear chromium assembly was found to activate theinsulin receptor activity similar to that observed with the LMWCr.(“Synthetic Multinuclear Chromium Assembly Activates Insulin ReceptorKinase Activity: Functional Model for Low-Molecular-WeightChromium-Binding Substance”, C. M. Davis et al, Inorg. Chem.,36:5316(1997))

The recognition that yet an unidentified complex of chromium (III) andorganic ligand(s) is responsible for modulating carbohydrate and lipidmetabolism has generated significant interest in developing novelchromium containing compounds for use in human and animal nutrition.Numerous patents have been issued describing compounds that containchromium bound to a variety of ligands. In 1975 a patent was issued toone of the inventors on this application disclosing 1:1 and 1:2Chromium, Alpha Amino Acid Complex Salts (U.S. Pat. No. 3,925,433).These complex salts exist as ion pairs in which the cation is composedof a complex of the chromium (III) ion with one or two molecules of analpha amino acid. The cation carries either a 1+ or a 2+ depending onthe number of amino acid molecules forming the complex. The counter ion(anion) may be chloride, sulfate or acid sulfate. Essential metalcomplexes of L-methionine, including 1:1 chromium-L-methionine complexesare disclosed in U.S. Pat. No. 5,278,329. Metal complexes of amino acidsobtained by hydrolysis of proteins, including chromium-amino acidcomplexes are described in U.S. Pat. No. 5,698,724.

A method for obtaining concentrated glucose tolerance factor fromBrewer's yeast was described in U.S. Pat. No. 4,343,905 issued in 1982.Other patents were issued since describing methods for obtaining yeastor yeast derivatives possessing biological activities in modulatingcarbohydrate or lipid metabolism, e.g. U.S. Pat. Nos. 4,348,483;6,140,107; 6,159,466 and 6,248,323.

The use of the previously known compound, Chromium Acetylacetonate as adietary supplement and pharmaceutical agent is described in U.S. Pat.No. 4,571,391. This water insoluble compound is heat stable, very stableto acids and slightly basic pH solutions. Chromium acetylacetonate isreported to be rapidly absorbed from the gastrointestinal tract afteroral administration and is effective in potentiating insulin effects onglucose metabolism.

Dietary supplementation with essential metal picolinate, includingchromium picolinate was first disclosed in U.S. Pat. No. 4,315,927 thatwas reissued on Jul. 7, 1992 as Re 33,988. In U.S. Pat. No. 4,315,927the preparation of chromium picolinate was described (Example 4). In Re33,988 specific claims are made to cover picolinate complexes ofchromium, cobalt, copper and manganese in addition to zinc and ferrousthat were covered in U.S. Pat. No. 4,315,927. A method for producingchromium picolinate complex is described in U.S. Pat. No. 5,677,461. Theuses of chromium picolinate in the treatment and prevention of variousdiseases are disclosed in a number of patents including U.S. Pat. Nos.5,087,623; 5,087,624; 5,175,156 and 6,329,361 B1. Compositionscontaining chromium picolinate and the uses of these compositions aredescribed in U.S. Pat. Nos. 5,614,553; 5,929,066; 6,093,711; 6,136,317;6,143,301; 6,251,888 B1 and 6,251,889 B1.

Chromium nicotinate, described as “GTF Chromium Material” and methodsfor its preparation were disclosed in U.S. Pat. Nos. 4,923,855 and5,194,615. The use of chromium nicotinate for lowering blood lipidlevels is described in U.S. Pat. No. 4,954,492. Compositions containingchromium nicotinate and their uses are disclosed in several patentsincluding U.S. Pat. Nos. 5,905,075; 5,948,772; 5,980,905; 6,100,250;6,100,251 and 6,323,192.

Pharmaceutical insulin-potentiating Cr (III) complexes possessingGTF-like activity are disclosed in U.S. Pat. No. 5,266,560. Thesecomplexes are composed of Cr (III), nicotinic acid or one of itsderivatives and glutathione (a peptide containing L-glutamic acid,L-cysteine and glycine). The insulin potentiating activity of thesecomplexes on glucose transport in isolated adipocytes in vitro isdescribed and compared to that of similar complexes previously reportedin the literature.

The use of metal proprionates, including chromium proprionate isdisclosed in U.S. Pat. Nos. 5,707,679 and 6,303,158 B1. A compositioncontaining chromium salts of short chain fatty acids and its use inanimal nutrition is described in U.S. Pat. No. 5,846,581. Methods forproducing metal carboxylate for use as animal feed supplements aredescribed in U.S. Pat. Nos. 5,591,878 and 5,795,615.

Bioavailable chelates of creatine and essential metals, includingchromium are described in U.S. Pat. No. 6,114,379. This patent claims acreatine-chromium complexes containing from 1-3 equivalents of theligand for each chromium ion.

The use as a nutritional supplement or in the treatment of medicalconditions of a previously known ti-nuclear chromium (III) complex isdescribed in U.S. Pat. Nos. 6,149,948 and 6,197,816 B1. The complex isrepresented by the formula [Cr₃O(O₂CCH₂CH₃)₆(H₂O)₃]⁺. The biologicaleffects of the complex on a number of enzymes involved in carbohydrateand lipid metabolism are described in these patents. A method for theisolation of bovine low-molecular weight Cr-binding substance and itsuse are described in U.S. Pat. No. 5,872,102. This substance enhancedthe insulin-activated uptake of glucose by rat adipocyts and activatedrat adipocytic membrane tyrosine kinase and phosphotyrosine phosphataseactivities.

Several shortcomings have been identified that limit the effectivenessof the various chromium complexes described in the literature. Chromiumpicolinate is the most popular of the commercially available chromiumcomplexes. However this compound has limited water solubility and somerecent studies questioned its safety. Although the lack of toxicity ofchromium chloride and chromium picolinate has been demonstrated in rats(“Lack of Toxicity of Chromium Chloride and Chromium Picolinate inRats”, Anderson et al, J. Amer. Coll. Nutr. 16: 273(1997), recentstudies reported that chromium picolinate cleaves DNA and produceschromosome damage in Chinese hamster ovary cells. (“The NutritionalSupplement Chromium (III) Tris (picolinate) Cleaves DNA”, J. K.Speetjens et al, Chem. Res. Toxicol. 12:483(1999) & “Chromium (III)picolinate produces chromosome damage in Chinese hamster ovary cells”,D. M. Stearns, FASEB J., 9:1643(1995)) A study of the in vivodistribution of chromium(III) picolinate in rats concluded that theshort lifetime of this compound in vivo minimizes the potential toxiceffects of this dietary supplement.(“In Vivo Distribution of Chromiumfrom Chromium Picolinate in Rats and Implications for the Safety of theDietary Supplement”, D. D. D. Hepburn and J. B. Vincent, Chem. Res.Toxicol., 15:93(2002)). For these reasons, it is clear that analternative source of dietary chromium that is soluble, bioavailable,efficacious and safe is needed.

It is a primary objective of this invention to fulfill the abovedescribed need.

It is another objective of this invention is to provide novel 1:3complexes of chromium (III) and alpha amino acids for use as nutritionalsupplement for humans and domesticated animals.

A still further objective of the invention is to provide methods forpreparation of these novel complexes.

Yet another objective is to provide and describe the desirable effectsof these complexes on animal performance.

An another objective of the invention is to demonstrate the lack oftoxicity of the novel complexes in laboratory animals.

The structures here are 1:3 complexes of chromium (III) and alpha aminoacids. The structure and properties of most of the availablenutritionally relevant chromium complexes have been previously studied.For example, the mononuclear and binuclear complexes of chromium (III)picolinate have been synthesized and their structures were determined byx-ray crystallography. The reaction of chromium (III) chloride withpicolinic acid in water at a pH<4.0 produced the mononuclear complex inwhich the ratio of metal to amino acid is 1:3 (chromium tri-picolinate).However, if the pH of the solution was>4.0, the binuclear complex wasformed. The ratio of chromium to amino acid in the binuclear complex is1:2. (“Mononuclear and Binuclear Chromium (III) Picolinate Complexes”,D. M. Stearns and W. H. Armstrong, Inorg. Chem., 31:5178(1992)).

The composition and biological activity of chromium complexes ofpicolinic acid and nicotinic acid have also been studied. The chromiumcomplexes formed with these pyridine carboxylic acids are differentbecause of the differences in the structure of the two compounds.Nicotinic acid is not an alpha amino acid and hence serves as amono-dentate ligand. It binds with chromium through the carboxylateanion and forms in and tri-nuclear complexes. Two complexes were formedbetween chromium and nicotinic acid, the 1:1 and 1:2. Neither complexhad biological activity in the battery of tests used in this studyexcept that the chromium dinicotinate potentiated insulin activity inrat isolated adipose tissue. Picolinic acid on the other hand is analpha amino acid and serves as a di-dentate ligand. It binds with thechromium ion through the pyridine nitrogen and carboxyl oxygen to form astable five-member ring. Three different complexes were obtained when asolution of chromium chloride was treated with picolinic acid dependingon the ratio of picolinic acid to chromium in the reaction mixture. Theaddition of one or two molar equivalents of picolinic acid to thechromium chloride solution caused a change in the color of the solution.Adjusting the solution to pH 7.4 with sodium hydroxide resulted in theprecipitation of the complexes. These complexes were found to behomogenous by High Performance Liquid Chromatography (HPLC). When onemolar equivalent of picolinic acid was used the product had thestructure Cr Pic (H₂O)₂(OH)₂. The precipitate obtained when two molarequivalents were used had the structure Cr(Pic)₂(H₂O)(OH).(H₂O). Sincethese complexes were formed at pH>4 they are most likely the binuclearcomplexes. Neither of the two complexes had biological activity. Theaddition of three molar equivalents of picolinic acid to a solution ofchromium chloride in water results in the formation of a red solid thatprecipitated from solution. This precipitate was found to be homogenousby HPLC. Analysis of the precipitate indicated that it is the chromiumtri-picolinate monohydrate, Cr (Pic)₃.H₂O. This material is most likelythe mononuclear complex. This complex increased glucose uptake by ratskeletal muscle cultures in vitro. Addition of the complex to rat dietproduced significant decrease in plasma glucose and prevented glycationof hemoglobin. Dietary supplementation of the chromium tripicolinate inHumans resulted in a significant increase in lean body mass in bothmales and females. (“Composition and Biological Activity ofChromium-Pyridine Carboxylate complexes”, G W Evans and D J Pouchnik, J.Inorg. Biochem., 49:177(1993))

It can therefore be seen that all of the structures currently availablediffer from the chromium compounds of the present invention which are adifferent empirical formula and a different stereochemistry.

SUMMARY OF THE INVENTION

This invention relates to the preparation of novel 1:3 chromium (III)complexes. These complexes contain chromium in the oxidation state plus3. The chromium in the complex is bound with three molecules of an alphaamino acid. In contrast to known neutral chromium complexes that arepractically insoluble in polar solvents, the novel complexes describedin this patent are readily soluble in polar solvents such as water andmethanol. The complexes are stable in acidic and basic solutions. Thesewater-soluble complexes are useful sources of readily bioavailablechromium when added to diets. The use of these complexes as feedadditives in animal nutrition improves animal performance. The complexesdid not produce toxicity when fed to laboratory rats at high doses.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

Chromium exists in several oxidation states but the most stable andimportant state is Cr(III). In its most stable state, trivalent chromiumhas a coordination number of six (6). The hexacoordinated trivalentchromium forms octahedral complexes with a variety of ligands. Thesecomplexes are characterized by their relative kinetic inertness inaqueous solutions. The half-life of the ligand-displacement reaction ofmany of these complexes is several hours. Because of this kineticinertness, many complexes can be isolated as solids and are stable inaqueous solution for relatively long times, even under conditions wherethey are thermodynamically unstable.

The present invention involves the design, synthesis and evaluation ofnovel safe and effective chromium-amino acid complexes. Features thatimpart biological activity to metal-amino acid complexes include watersolubility, stability of the complex at the pHs of the GI contents,absorbability of the complex and the ability of the complex toparticipate in biochemical reactions. The safety of metal-amino acidcomplexes is enhanced by the use of natural amino acids and by improvingits bioavailability to minimize the amount of metal added to the feed tomeet the nutritional requirements of animals.

In U.S. Pat. No. 3,925,433 1:1 and 1:2 chromium-alpha amino acidscomplex salts are described. Although these complexes provide importantnutritional advances over inorganic sources of chromium, they sufferfrom several shortcomings. Mixing a solution of chromium chloride withone, two or three molar equivalents of an alpha amino acid results inthe formation of clear green solutions. The pHs of these solutions were0.932, 1.324 and 1.627, respectively. Adjusting these solutions to pH 7by the careful addition of sodium hydroxide or sodium carbonate solutionresulted in the precipitation of chromium compounds indicating thatthese complexes may not be sufficiently stable at range of pH valuesthat may be present in the gastrointestinal tract. When a solution ofthe chromium-alpha amino acid complex (1:3) was treated with threeequivalents of sodium hydroxide, a purple precipitate was formed. Thisprecipitate was practically insoluble in water, dilute acids and bases,methyl alcohol, ethyl alcohol, isopropyl alcohol and ethyl acetate.Elemental analysis and examination of its FTIR indicated that it is amixture of the neutral complex of chromium-amino acid (1:3) togetherwith some poly-nuclear chromium-amino acid complexes. The lack ofsolubility of this complex and its uncertain composition suggested thatit is unlikely to be of nutritional value.

The novel complexes described in this invention are represented bystructure 1 and are salts of the 1:3 chromium-amino acid complexes.These complexes exist as ion pairs in which the monovalent cation iscomposed of trivalent chromium complexed with three molecules of analpha amino acid. One of the amino acid molecules retains itszwitterionic character to impart a net positive charge on the complex.The carboxylate group of this amino acid forms two bonds with thechromium (III) ion forming a strained four-member ring that satisfiestwo of the chromium six coordination sites. The other two alpha aminoacid molecules bind to chromium through the alpha amino and carboxylgroups to form five member rings. This will satisfy all the sixcoordination bonds of chromium (III). The anion of the ion pair referredto here as “X” may be a monovalent anion such as chloride or a divalentanion such as sulfate. “R” is the organic moiety of an alpha amino acid.It can be derived from arginine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, threonine, tryptophan valine, glycine, etc.While glycine is not as an essential amino acid, it is also a preferredalpha amino acid in that it is readily available and can easily beutilized for synthesis of the complex salts of this invention. The twomost preferred natural alpha amino acids are glycine and methionine. Forglycine R represents hydrogen, and for methionine R represents thefollowing: CH₃—SC—H₂—CH₂—.

These complexes can be prepared by using a simple and practical method.A solution of chromium chloride in water is heated to 90-95° C. Thesolution is usually dark green in color. The amino acid (3 molarequivalents) is carefully added and the heating is continued. The colorof the solution slowly changes to dark blue-green. The solution iscooled to about 40° C. and sodium hydroxide solution is slowly andcarefully added to adjust the solution to pH 3.9-4.0. Two molarequivalents of sodium hydroxide are required for the pH adjustment. Thecolor of the solution turns to dark purple. Evaporation of the liquidprovides a solid that is composed of the desired product and sodiumchloride. The product can be separated from sodium chloride byextraction with methanol or ethanol. Alternatively, product may beseparated from sodium chloride by chromatography on a suitable sizeexclusion resin.

The product obtained using the method described above has several uniqueproperties. It exists as a stable solid. It is dark purple in color. Theproduct is readily soluble in water and methanol, soluble in ethanol,sparingly soluble in isopropyl alcohol and insoluble in ethyl acetate. A0.1 molar solution of the product in water has a pH of 4.078. The UV/Visspectrum of the solution has absorption maxima at 400 nm (molarabsorptivity, 44.08) and at 541 nm (molar absorptivity, 50.60). Incontrast, the UV/Vis spectrum of chromium chloride has absorption maximaat 429 nm (molar absorptivity, 18.10) and at 608 nm (molar absorptivity,14.43). The addition of as many as 20 molar equivalents of a 0.1 molarsolution of sodium bicarbonate changed the pH of the solution to 8.097but no precipitate was formed. In one experiment as much as 80 molarequivalents of the 0.1 molar solution of sodium bicarbonate was added.The pH of the mixture was 8.354 but no precipitate of chromium compoundswere formed. In contrast, the addition of slightly more than 2equivalents of 0.1 molar sodium bicarbonate to a 0.1M solution ofchromium chloride resulted in the formation of a voluminous precipitatebut the pH of the mixture was only 4.356. Adjusting the 0.1 molarsolution of the complex to about pH 1.0 by the addition of 20 molarequivalents of hydrochloric acid did not decompose the complex asmanifested by no change in the UV/is spectrum of the solution.

The solid complex obtained by using the method described above ishomogenous by High Performance Liquid Chromatography. Analysis of amethanol solution by using a size exclusion column indicated thepresence of a single component. Detection of the peak at two differentwavelengths, one to detect chromium and the second to detect the aminoacid indicated that both eluted in the single peak. Analysis of amethanol solution of chromium chloride indicated that it eluted asseveral peaks, none was similar to that of the complex. Further themajor peak of chromium chloride had a shorter retention time than thecomplex indicating a larger molecular size.

Complexes with similar physico-chemical properties were obtained usingdifferent alpha amino acids. Elemental analysis of the complexes gaveresults consistent with the proposed structure. The FTIR spectrum of thecomplex is different than that of the amino acid used for forming thecomplex and is similar to that of amino acid complexes of other metals.

The complex formed between chromium (III) and L-methionine was tested asa nutritional source of chromium in dairy cows and was found to improveperformance. Supplementing the diet with chromium tri-L-methioninehydrochloride did not affect the intake of dry matter in cows butincreased milk production. The toxicity of the complex in rats wasexamined and found to be non-toxic in the doses used.

EXAMPLE 1

Preparation of Chromium (III) Tri-methionate Hydrochloride:

Water (550 ml) was placed in a 2000 ml-beaker. Chromium chloridehexahydrate (79.959 g, 0.3 moles) was added. The mixture was heated withstirring to boiling. L-Methionine (134.306 g, 0.9 moles) was added. Themixture was heated with stirring until the solids completely dissolved.Heating with stirring was continued for additional 30 minutes. Thesolution turned from dark green to dark blue-green. The solution wascooled to 30° C. Sodium hydroxide (23.316 g, 0.5829 moles) was dissolvedin 100 ml water and the solution was cooled to 30° C. The sodiumhydroxide solution was added dropwise to the chromium-methioninesolution with stirring. The solution turned from dark blue-green to darkpurple. The solution was evaporated to dryness under reduced pressure.The residue was extracted with methanol to leave a white crystallinesolid. The methanol extract was evaporated to dryness to give a darkpurple crystalline solid (161.352 g, yield 100.91%).

FTIR of product in a potassium Bromide pellet showed absorptions at:3421.5(s), 2916.2(s), 1635.5(s), 1508.2(m), 1438.8(m), 1338.8(m),1338.5(s), 1272.9(w), 1242.1(w) and 1145.6(m) cm⁻¹. (s=Strong, m=Medium,w=Weak)

The visible spectrum of a 0.01 M solution in water had two maxima at 400nm (molar absorptivity, 44.08) and at 541 nm (molar absorptivity,50.60).

The pH of a 0.1 molar solution in water was 4.078. A 10 ml portion wasdiluted with 200 ml of a 0.1 molar solution of sodium bicarbonate. Noprecipitate was formed. The solution had a pH of 8.097. An additional600 ml of 0.1 molar sodium bicarbonate solution was added. Noprecipitate was formed and the pH of the solution was 8.354.

A solution of the complex in methanol containing the equivalent of0.9555 mg/ml chromium was analyzed by HPLC on a 60A Macrosphere GPCcolumn (Alltech Associates, Inc.) using methanol as mobile phase at arate of 0.5 ml/min and a UV/Vis detector at 407 nm. A single peak withretention time of 6.48 min. was obtained. When a solution of chromiumchloride hexahydrate containing the equivalent of 0.4666 mg/ml chromiumwas analyzed under the same condition a peak eluted with retention timeof 6.67 min. The longer retention time indicates that the chromiumcomplex has a larger molecular size than chromium chloride.

The HPLC analysis was repeated except for setting the detector to 210 nmto detect the amino acid. The sensitivity of detection was more than 100folds greater than that at 405 nm and it was necessary to dilute thesample to contain 0.009555 mg/ml chromium. This diluted sample gave asingle peak with retention time of 6.19 min at 407 nm. At 210 nmmultiple minor peaks were observed with the major peak having retentiontime of 6.19 min. Analysis of a sample of L-methionine hydrochlorideunder the same condition with the detector set at 210 nm indicated thepresence of several minor peaks in addition to the major peak at 7.6min. These results indicate that the complex migrated on the columnintact and that its molecular size is larger than that of chromiumchloride and L-methionine hydrochloride.

EXAMPLE 2

Preparation of Chromium (III) Tri-leucinate Hydrochloride:

Water (150 ml) was placed in a 600 ml-beaker. Chromium chloridehexahydrate (13.325 g, 0.05 moles) was added. The mixture was heatedwith stirring to boiling. L-Leucine (19.685 g, 0.15 moles) was added.The mixture was heated with stirring until the solids completelydissolved. Heating with stirring was continued for additional 30minutes. The solution turned from dark green to dark blue-green. Thesolution was cooled to 30° C. Sodium hydroxide (4.014 g, 0.10 moles) wasdissolved in 20 ml water and the solution was cooled to 30° C. Thesodium hydroxide solution was added dropwise to the chromium-leucinesolution with stirring. The solution turned from dark blue-green to darkpurple. The solution was evaporated to dryness under reduced pressure.The residue was extracted with methanol to leave a white crystallinesolid. The methanol extract was evaporated to dryness to give a darkpurple crystalline solid (28.363 g, Theory 23.948 yield 118.44%indicating that the product contains residual sodium chloride).

FTIR of product in a potassium Bromide pellet showed absorptions at:3425.3(m), 2916.2(s), 1635.5(s), 1508.2(m), 1438.8(m), 1384.8.8(m),1338.5(s), 1272.9(w), 1242.1(w) and 1141.8(m) cm⁻¹ (s=Strong, m=Medium,w=Weak)

The visible spectrum of a 0.01 M solution in water had two maxima at 406nm (molar absorptivity, 38.79) and at 545 nm (molar absorptivity,42.81).

The pH of a 0.1 molar solution in water was 3.996. A 10 ml portion wasdiluted with 200 ml of a 0.1 molar solution of sodium bicarbonate. Noprecipitate was formed. The solution had a pH of 7.987. The visiblespectrum of the solution had two maxima at 409 m (molar absorptivity,47.46) and at 558 nm (molar absorptivity, 47.67).

EXAMPLE 3

Preparation of Chromium Tri-methionate Hydrochloride Premix (0.1%Chromium):

A 100-ml of distilled water was measured into a 400-ml beaker. ChromiumChloride Hexahydrate (6.672 g, 0.0251 moles) was added and the mixturewas heated with stirring until the solid was completely dissolved.L-Methionine (11.201 g, 0.0751 moles) was added and the mixture washeated with stirring. The color of the solution changed from dark greento blue-green. Heating at 90-95° C. was continued for additional 60 min.The solution was cooled to 30° C. A cooled solution of sodium hydroxide(1.967 g, 0.0492 moles) in 50 ml of water was added dropwise withstirring. The color of solution changed to dark purple. The solution wasevaporated under reduced pressure. The residue was dissolved in 100 mlof methanol and the solution was added to 1000 g of a carrier. Themixture was placed in an oven at 60 min for 24 hrs. A sample of thedried premix was analyzed as described in Example 1 by UV/Vis,colorimetry and HPLC. The premix was used in a feeding trial in swine.

EXAMPLE 4

Preparation of Chromium Trimethionate Solution (1.75% Chromium):

A 1500 ml of water was measured into a 4-1 beaker. Chromium ChlorideHexahydrate (390.993 g, 1.4675 moles) was added and the mixture washeated with stirring until the solids completely dissolved. Heating withstirring was continued for additional 1 hr to completely hydrate thechromium salt. L-methionine (1094.845 g, 7.338 moles) was added and themixture was heated with stirring until all the solids dissolved. Heatingwith stirring was continued for 1 hr. The color of the solution changedto dark purple. The solution was completed to 4 liters with distilledwater.

The pH of the solution was 2.336. It contained 25.61% methionine and1.86% chromium.

The visible spectrum of a 0.01 M solution in water had two maxima at 416nm (molar absorptivity, 102.5) and at 579.5 nm (molar absorptivity,101.9).

The pH of a 0.1 molar solution in water was 2.285. A 10 ml portion wasdiluted with 200 ml of a 0.1 molar solution of sodium bicarbonate. Noprecipitate was formed. The solution had a pH of 7.546. The visiblespectrum of the solution had two maxima at 409.5 nm (molar absorptivity,43.89) and at 558 nm (molar absorptivity, 46.83).

EXAMPLE 5

Effects of Chromium Tri-L-Methionate Hydrochloride on Performance ofDairy Cows:

The effects of supplementation with chromium in the form of chromiumtri-L-methionine hydrochloride on performance of periparturient dairycows were studied. Seventy-two cows were used to determine whether milkproduction and dry matter intake were affected by chromiumtri-L-methionine hydrochloride supplementation of the diet during theperiparturient period. Cows were fed either a diet high in nonforagefiber sources or high in non-fiber carbohydrates from day 21 beforeexpected parturition until parturition and then fed a common lactatingdiet. Chromium tri-L-methionine hydrochloride was supplemented oncedaily via gelcap at doses of 0, 0.03, or 0.06 mg Cr/kg of metabolic bodyweight. Chromium supplementation began on day 21 before expectedparturition and continued until day 28 postpartum. Cows were milkedafter calving according to established procedures. Feed intake wasrecorded daily for each cow throughout the experiment. Samples of thediet were obtained weekly and the dry matter contents were determined.Individual milk weights were recorded at each milking during thelactating phase of the experiment. Milk samples were taken from allmilkings during one 24-hours period each week, composited based on theamount of milk produced at each milking, and analyzed for fat, protein,lactose, and total solids.

Supplementing diets with chromium tri-L-methionine hydrochloride did notaffect dry matter intake in cows. However, chromium supplementationtended to increase milk yield (P<0.13,Table 1).

TABLE 1 Chromium tri-L-Methionine HCl mg/kg BW^(.75) 0.00 0.03 0.06Prepartum Dry Matter Intake 13.6 13.9 13.7 kg/day Postpartum Dry MatterIntake 17.9 18.9 19.4 kg/day Milk Production kg/day 40.4 40.6 42.8 MilkFat kg/day 1.73 1.76 1.81 % Milk Fat 4.43 4.41 4.33 Milk Protein kg/day1.28 1.33 1.31 % Milk Protein 3.32 3.37 3.15 Milk Lactose kg/day 1.861.91 1.98 % Milk Lactose 4.63 4.69 4.61 Milk Solids kg/day 5.27 5.375.51 % Milk Solids 13.38 13.39 13.03The chromium (III) complexes of this example and the invention can beused with conventional inert nutritional carriers such as distillersfermentation solubles, feed grains, poultry and fish bi-products, meal,whey, natural salt, ground corn cobs, feathermeal, etc.

EXAMPLE 6

Toxicity of Chromium Tri-L-Methionine Hydrochloride in Rat:

The toxicity of Chromium-L-Methionine Hydrochloride was studied in therat following the administration of a single oral dose. Fifty 6 weeksold rats were used in the study (25 females and 25 males). Their bodyweight ranged from 130 to 220 g for males and 120-190 g for females. Theanimals were housed in polycarbonate cages over dust-free sawdustbedding in groups of five. The cages were placed in an air-conditionedroom at 22° C. and 55% relative humidity. Rats were fed pelletedcomplete diet ad libitum. Animals were fasted overnight before dosingand given food 3-4 hours after dosing. Animals given water ad libitum.Animals were assigned at random to one of five treatments. Two groups offive animals each, one group of males and one group of females wereassigned to each treatment. The treatments were control, 250 mg/kg, 500mg/kg, 1000 mg/kg and 2000 mg/kg doses. A solution of the compound inwater was administered in a single oral dose by gastric gavage. Animalswere maintained for a 14-day observation period after receiving thedose. Surviving animals were killed on day 14. The weight of animalswere recorded immediately before treatment, twice weekly for the studyperiod and at death.

No animals died during the study and no treatment-related clinical signswere seen in the animals given the compound at any of the dose levels.Furthermore there were no macroscopic abnormalities in animalsnecropsied on day 14 after the oral dose. However, a 5% decrease in bodyweight gain was observed in male rats given the 2000 mg/kg dose. Thisdose is 4000 fold the recommended dose for pigs. Also, a dose relateddecrease in food consumption compared to control was noted in groupstreated with the 1000 and 2000 mg/kg doses.

These results indicate that under these experimental conditions, theadministration of a single oral dose of chromium tri-methionine in ratdid not induce any toxicity at dose levels 1000 folds the recommendeddose in pigs. The administration of a dose 2000 folds the recommendeddose was associated with a decrease in food consumption, but was notassociated with a decrease in the gain in body weight. At the 2000 mg/kgdose, which is 4000 folds the recommended dose; the decrease in foodconsumption was associated with a slight decrease in body weight gain.

1. A chromium complex of the formula:

wherein “R” is an organic moiety normally present in a natural aminoacid and “X” is a water soluble anion.
 2. The complex of claim 1 wherein“X” is monovalent.
 3. The complex of claim 1 wherein “X” is selectedfrom the group consisting of chloride, bromide, iodide, sulphate,phosphate, acetate and propionate.
 4. The complex of claim 1 wherein “R”is selected so that the natural amino acid is one selected from thegroup consisting of arginine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, threonine, tryptophan, valine and glycine. 5.A nutritional composition of enhanced bioavailability for livestock,comprising a chromium complex of the formula:

in combination with an animal nutrition carrier.
 6. The complex of claim5 wherein “X” is monovalent.
 7. The complex of claim 5 wherein “X” isselected from the group consisting of chloride, bromide, iodide,sulphate, phosphate, acetate and propionate.
 8. The complex of claim 5wherein “R” is selected so that the natural amino acid is one selectedfrom the group consisting of arginine, histidine, isoleucine, leucine,lysine, methionine, phenylalanine, threonine, tryptophan, valine andglycine.
 9. The composition of claim 5 wherein the carrier is selectedfrom the inert animal nutrition carrier group of natural salt, groundcorn cobs, corn meal, and feathermeal.
 10. A nutritional composition ofenhanced bioavailability for livestock, comprising a chromium complex ofthe formula:

wherein “R” is an organic moiety normally present in a natural aminoacid and “X” is a water soluble anion, all in a liquid form carrier. 11.The complex of claim 10 wherein “X” is monovalent.
 12. The complex ofclaim 10 wherein “X” is selected from the group consisting of chloride,bromide, iodide, sulfate, phosphate, acetate and propionate.
 13. Thecomplex of claim 10 wherein “R” is selected so that the natural aminoacid is one selected from the group consisting of arginine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, threonine,tryptophan, valine and glycine.
 14. The composition of claim 10 whereinthe solvent is selected from water, ethanol, molasses or any othersuitable solvent or mixture of suitable solvents.